Time-Resolved Interactome Profiling Reveals Temporal Dynamics of Protein Quality Control Pathways in Secretory Protein Biogenesis

Core Concepts

Time-resolved interactome profiling (TRIP) can identify temporal changes in protein-protein interactions that govern the proteostasis network and regulate the biogenesis of secretory proteins, providing insights into the mechanisms underlying protein folding diseases.

Abstract

The content describes the development and application of a novel method called time-resolved interactome profiling (TRIP) to map the temporal dynamics of protein-protein interactions involved in the protein quality control (PQC) network. The key highlights are:

TRIP utilizes pulsed unnatural amino acid labeling and a two-stage enrichment strategy to capture and quantify the interactions between a client protein (thyroglobulin, Tg) and its interacting partners over time.

TRIP revealed distinct temporal profiles of Tg interactions with components of the Hsp70/90 chaperone system, disulfide/redox processing, glycan processing, and protein degradation pathways between wild-type Tg and disease-associated Tg mutants.

The altered temporal interactions of mutant Tg with PQC components suggest dysregulation of protein folding, trafficking, and degradation pathways, which contribute to the secretion defects observed in the protein folding disease congenital hypothyroidism.

An siRNA screen identified VCP and TEX264 as key regulators of Tg processing, and pharmacological inhibition of VCP selectively rescued the secretion of the mutant Tg by remodeling its interactome.

The study demonstrates the power of TRIP to delineate the temporal coordination of the proteostasis network and its dysregulation in protein folding diseases, paving the way for targeted therapeutic interventions.

Stats

"Tg mutants are characterized by both discrete changes with select PN components and broad temporal alterations across Hsp70/90 and disulfide/redox processing pathways."
"VCP silencing increased both A2234D and C1264R Tg-NLuc secretion, while HERPUD1, TEX264 and RTN3 silencing selectively increased C1264R secretion."
"ML-240 treatment led to a 10-fold increase in C1264R Tg secretion at the 4 hr time point with no significant change in C1264R Tg degradation."

Quotes

"TRIP has allowed identification and resolution of unique temporal changes in Tg interactions with glycan processing components including STT3B, CANX, CALR, and UGGT1, while contrasting WT to mutant Tg variants."
"Identification of these receptors establishes a direct link from Tg processing to ER-phagy or ERLAD degradation mechanisms."
"TRIP was capable of resolving subtle temporal interaction changes, for example with CANX, ERP29, and VCP, which were otherwise masked in steady-state interactomics data."

Key Insights Distilled From

Time-Resolved Interactome Profiling Deconvolutes Secretory Protein Quality Control Dynamics

by Wright,M. T.... at www.biorxiv.org 09-05-2022

https://www.biorxiv.org/content/10.1101/2022.09.04.506558v1

Deeper Inquiries

How can the TRIP methodology be further optimized to improve the temporal resolution and quantification of protein-protein interactions, especially for components involved in translation, trafficking, and degradation?

The TRIP methodology can be optimized in several ways to enhance the temporal resolution and quantification of protein-protein interactions, particularly for components involved in translation, trafficking, and degradation.

Improved Labeling Strategies: Utilizing more advanced labeling strategies, such as metabolic labeling with stable isotopes or incorporating multiple labeling time points, can provide a more comprehensive temporal profile of protein interactions.

Enhanced Mass Spectrometry Techniques: Implementing cutting-edge mass spectrometry techniques, such as data-independent acquisition (DIA) or parallel reaction monitoring (PRM), can improve the sensitivity and accuracy of protein quantification, especially for low abundance proteins involved in translation and trafficking.

Integration of Live Cell Imaging: Combining TRIP with live cell imaging techniques, such as fluorescence resonance energy transfer (FRET) or proximity ligation assays, can offer real-time visualization of protein interactions, providing dynamic insights into the temporal regulation of these interactions.

Incorporation of Crosslinking Strategies: Introducing crosslinking agents at specific time points during the TRIP workflow can stabilize transient protein-protein interactions, allowing for the capture of short-lived interactions involved in translation, trafficking, and degradation processes.

Integration of Computational Modeling: Implementing computational modeling approaches, such as network analysis or kinetic modeling, can help in predicting and validating the temporal dynamics of protein interactions, offering a more holistic view of the proteostasis network.

How can the potential therapeutic implications of modulating the interactions between mutant secretory proteins and the proteostasis network components identified in this study be translated to other protein folding diseases?

The potential therapeutic implications of modulating interactions between mutant secretory proteins and the proteostasis network components identified in this study can be translated to other protein folding diseases through the following strategies:

Targeted Drug Development: Utilizing the identified proteostasis network components as therapeutic targets, novel drugs can be developed to modulate protein interactions and improve the folding and secretion of mutant proteins in various protein folding diseases.

Precision Medicine Approaches: Implementing precision medicine approaches, such as patient-specific modulation of proteostasis network components based on the individual's genetic profile and disease characteristics, can enhance the efficacy of therapeutic interventions in diverse protein folding disorders.

Combination Therapies: Developing combination therapies that target multiple components of the proteostasis network simultaneously can provide synergistic effects in enhancing protein folding and secretion, offering a more comprehensive treatment strategy for protein folding diseases.

Clinical Trials and Validation Studies: Conducting clinical trials and validation studies to assess the efficacy and safety of modulating proteostasis network components in diverse protein folding diseases can validate the therapeutic potential of these interventions and pave the way for clinical implementation.

What are the broader cellular functions and regulatory mechanisms of the ER-phagy receptors, such as TEX264, in maintaining proteostasis and how do they interface with other degradation pathways?

The ER-phagy receptors, including TEX264, play crucial roles in maintaining proteostasis through the selective degradation of ER components and misfolded proteins. These receptors interface with other degradation pathways in the following ways:

ER-Phagy and ERAD Crosstalk: ER-phagy receptors, such as TEX264, interact with components of the ER-associated degradation (ERAD) pathway to facilitate the clearance of misfolded proteins that are resistant to ERAD, ensuring efficient proteostasis maintenance.

Lysosomal Degradation Pathways: ER-phagy receptors target specific ER components for lysosomal degradation, promoting the turnover of damaged organelles and maintaining cellular homeostasis through the autophagic clearance of ER fragments.

Protein Quality Control: ER-phagy receptors contribute to protein quality control by selectively recognizing and sequestering misfolded proteins within the ER for degradation, preventing the accumulation of toxic protein aggregates and maintaining cellular proteostasis.

Integration with UPR Signaling: ER-phagy receptors are intricately linked to the unfolded protein response (UPR) signaling pathway, coordinating the degradation of unfolded proteins and ER stress mitigation to ensure cellular adaptation to proteotoxic stress conditions.

Regulation of ER Dynamics: ER-phagy receptors, such as TEX264, regulate ER dynamics by targeting specific ER regions or components for degradation, contributing to ER remodeling and turnover processes essential for cellular function and adaptation to stress conditions.